The squamous cell carcinoma antigen (SCCA) serves as a diagnostic serum marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, and head and neck (HNSCC). For certain tumors, SCCA also serves as a prognostic indicator. Elevated pretreatment SCCA levels portend a bad outcome in stage IB and IIA cervical carcinomas. Moreover, a persistent increase of SCCA after radiation therapy (RT) is the most important negative predictor of disease-free survival in HNSCC. Interestingly, RT induces a rapid increase in both the intracellular and extracellular amounts of SCCA synthesized by SCC cells. These findings suggest that between cellular SCCA content and radioresistance are not understood. To better understand the relationship of SCCA to the pathogenesis of SCC, we undertook a series of molecular and biochemical analyses. Genomic analysis of SCCA reveals the presence of two, tandemly arrayed genes, SCCA1 and SCCA2. The genes encode 390 amino acid proteins with high homology to members of the high molecular weight serine proteinase inhibitor (serpin) family. Although SCCA1 and SCCA2 are nearly identical in their amino acid sequence, biochemical analysis shows that they inhibit distinct classes of proteinases. SCCA1 paradoxically inhibits lysosomal cysteine proteinases, whereas SCCA2 inhibits chymotrypsin-like serine proteinases. Since cysteine and serine proteinases augment intracellular injury, especially that induced by post-irradiation oxidative stress, we hypothesize that increases in intracellular SCCA1 and/or SCCA2 content projects tumors from radiotherapy by blocking proteinase-mediated damage. The objective of this proposal is to test this hypothesis while gaining initial insight into the proteolytic cascades that, under certain intracellular conditions, may activate or even supersede caspase-mediated cell death pathways. The specific aims are to: 1) Determine directly whether the expression of SCCA 1/2 protects SCC cells from the lethal effects of ionizing irradiation, 2) identify the potential site of action of SCCA1 and SCCA2 by determining their precise subcellular localization in SCC tumor cell lines before and after irradiation, and 3) identify the cytosolic or nuclear targets of SCCA1/2 in irradiated SCC cells. These studies have broad implications by identifying proteinases from more than one mechanistic class that are capable of mediating radiation-induced injury and by ascribing a biologic activity to members of the serpin family whose function heretofore has been unrecognized. Finally, these studies may establish the presence of a novel intracellular defense mechanism for safeguarding against the consequences (e.g., lysosomal injury and proteinase release) of an overwhelmed antioxidant defense system.